The ultimate goal of transplantation is to achieve antigen-specific tolerance. Most attempts to do so involve the activation of T regulatory cells (Treg) that suppress the alloaggressive T cell response. We have recently obtained data that shows that the exogenous induction of heme oxygenase-1 (HO-1) facilitates tolerance induction by donor specific transfusions (DST) and thus may be useful as a complementary treatment to optimize the probability of achieving tolerance. HO-1 synergizes with DST in the successful induction of tolerance. We shall use induction of HO-1 or administration of carbon monoxide (CO) or biliverdin, products of heme degradation by HO-1, to promote induction of tolerance. In addition, we find that HO-1 activity is essential in the recipient for the induction of Treg-based tolerance in two models we have tested. Tolerance induced by blockade of CD40L (with MR-1) plus DST in BALB/c recipients of C57BL/6 hearts is highly effective in wild-type recipients but not effective in BALB/c hmox1-/- recipients, i.e. in which HO-1 has been globally deleted. Likewise, DST given on day -7 if HO&#1048577;1 activity in the recipient is blocked by zinc protoporphyrin IX can no longer induce tolerance. We wish to understand the action of HO-1 in the recipient that allows tolerance to be induced. We hypothesize that it is HO-1 expression in dendritic cells (DC) that is required to maintain those cells with an immature DC (iDC) phenotype so that the iDC can stimulate Treg that then function to yield tolerance. We do not rule out the needed expression of HO-1 in other cells. Our first specific aim includes in vitro and in vivo experiments to study the cell that must express HO-1 to get tolerance as well as to study the effects of HO-1 expression in DC with regard to phenotype and function of those cells, and whether carbon monoxide and biliverdin can substitute for HO-1 in these regards. Specific aims 2 and 4 attempt to elucidate the signaling pathways in DC in vitro and in recipients in vivo that account for the role HO-1 plays in guiding the phenotype of DC toward the immature state. To demonstrate the obligatory role of HO-1 in maintaining iDC status, we will perform confirmatory experiments using the conditional HO-1 knockout mouse, hmox1 loxp whereby HO-1 is deleted from DC using DC specific cre recombinase mice, thus generating hmox1 cd11c-cre lox mice. Finally in specific aim 3, we wish to capitalize on our observation that the combination of induced HO-1 expression with DST can be synergistic in terms of achieving tolerance by evaluating whether biliverdin or CO can substitute for HO-1 in tolerance induction. This would hopefully allow the combined use of CO or biliverdin with DST or some other tolerance-inducing regimen to develop a pre-clinical protocol.